The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The detection of biological and chemical agents of consequence for human health is an ongoing goal for various biotechnology platforms (1). Towards this end, nucleic acids that bind to small molecules in a highly specific manner, aptamers, have garnered interest for their ease of synthesis and quality control. A special class of aptamers, structure-switching aptamer beacons, contain intra-duplexes or inter-duplexes that are covalently linked to quencher and fluorescent probes. Upon binding of a target molecule, the proximity of the probes is disrupted, rendering the device fluorescent (2, 3).
These aptamer beacons have been generated via one of two general strategies. In the first approach, known aptamers are rationally modified by the addition of short sequence extensions. These additional bases serve to disrupt the aptamer's native fold and to introduce a fluorophore and quencher pair that can be attached covalently (4-7) (FIG. 1a) or by hybridization (8). In this latter example, both two-piece and three-piece aptamer beacons have been developed (FIG. 1b,c).
Second, structural-switching aptamers have been directly acquired through in vitro selection (9-12). Here, random nucleic acid libraries are typically immobilized on a support via hybridization (FIG. 1d). Following target addition, sequences that selectively bind a given target are released from the matrix if the binding event induces a structural rearrangement wherein an internal complementary sequence of the aptamer is preferentially bound. DNAs that are eluted in this fashion are PCR-amplified to seed a new, enriched library for use in subsequent rounds of selection. Recently, several steroid-binding aptamers were isolated from a DNA library that included a designed three-way junction containing eight variable bases (13)—a surprising result given the library contained less than 105 unique sequences. However, it is noted that even when using a small library such as this, the identification of functional aptamer beacons required 9 to 13 rounds of selection. Additionally, the selected molecules were not in and of themselves fluorescent sensors and had to be adapted and optimized for this purpose, further complicating the process of generating these sensors.
The present invention addresses the need for better methods for selection and identification of structural-switching aptamers or aptamer beacons.